In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. <br><br>Structural defects of the heterotrimeric type I collagen molecule are well known to cause the dominant bone disorder osteogenesis imperfecta. A severe recessive form of OI was first postulated in 1979. More recently, investigators have noted that some patients with clinical OI do not have defects detected in the type I collagen genes during sequencing. These patients without mutations in collagen can be divided into those who have abnormal collagen biochemistry and those with normal electrophoretic migration of the collagen chains. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen. Eight years ago the BEMB identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in proteins that interact with collagen cause the rare forms of OI. In the expanded nosology for OI, defects in CRTAP and LEPRE1 are designated as types VII (OMIM #610682) and VIII (OMIM #610915) OI, respectively. Among our LEPRE1-deficient patients, we identified a common mutant allele, IVS5+1G to T, which occurred in both African-Americans and West African families. This so-called West-African allele accounts for a third of the known LEPRE1 mutations, and has been found only in individuals of African descent. To our surprise, contemporary West Africans have a carrier frequency for this lethal recessive mutation of 1.5%! Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. Biochemically, both groups full overmodification of the helical prolines and lysines by prolyl 4-hydroxylase and lysly hydroxylase, indicating delayed folding of the collagen helix. We showed that mutual stabilization of CRTAP and P3H1 underlies the phenotypic and biochemical similarity of types VII and VIII OI. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry. We generated a CyPB KO mouse, which has reduced bone density and strength, but increased brittleness. Only 1-2% 3-hydroxyltion is detected in KO cells, showing the importance of CyPB to complex function. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports LH1 activity and in its absence there is significant reduction of hydroxylation of crosslinking residue K87. The decreased crosslink ratio alters fibril structure and reduces bone strength. Recessive mutations in FKBP10, which encodes FKBP65, cause type XI OI. We identified an FKBP10 mutation in Kuskokwim syndrome (KS), a recessive congenital contracture disorder among Yupil Eskinos in Alaska. The causative mutation deletes the highly conserved p.Tyr293 residue in FKBP65s third PPIase domain, substantially destabilizing the protein. FKBP65 supports LH2 function; absence of FKBP65 decreases hydroxylation of the telopeptide lysine important for collagen crosslinking. Most recently, we have delineated a muttion in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. We identified a patient with severe OI whose fibroblasts and osteoblasts secreted minimal amounts of PEDF and whose bone histology was typical of type VI OI, but whose serum PEDF was in the normal range. Whole exome sequencing revealed a de novo mutation in IFITM5 in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL. Both IFITM5 transcripts and BRTIL protein levels were normal in proband cells. However, SERPINF1 expression was minimal. Expression of type I collagen was similarly decreased in proband osteoblasts, and the pattern of osteoblast markers was consistent with a primary PEDF defect. Since this mutation in IFITM5 was causing bone-specific type VI OI, we compared these osteoblasts to osteoblasts with the type V OI-causing IFITM5 mutation at the 5:-end of the gene. In these cells we demonstrated increased SERPINF1 expression and PEDF secretion during osteoblast differentiation, connecting the two OI-causing genes in an important pathway under delineation. We also investigated the effect of the IFITM5 mutation on mineralization and differentiation by type V OI osteoblasts in culture. BRIL was shown to be stably expressed by these osteoblasts at normal levels. In support of a gain-of-function model, type V OI osteoblasts deposit increased mineralization in culture, and have increased expression of alkaline phosphatase, bone sialoprotein, osteopontin and osteocalcin. Only type I collagen shows decreased expression, secretion and matrix incorporation in these cells, similar to findings in cells with the BRIL S40L mutation. We also delineated a new gene causing a complex congenital syndrome that overlaps OI and ciliopathies. The syndrome is characterized by fetal lethality, severe hypomineralization of the skeleton with intrauterine fractures, and multiple anomalies of brain, lung and kidney. Along with colleagues from Ghent the defect in this syndrome was traced to the gene TAPT1, encoding a transmembrane protein critical to anterior posterior transformation. Our studies localized normal TAPT1 to the centrosome/ciliary body, but mutant TAPT1 mislocalizes to the cytoplasm and disrupts both Golgi trafficking of collagen and primary cilium formation.